Abstract
The concentration level of urinary 8-hydroxy-2′-deoxyguanosine (8-OHdG), an oxidative stress biomarker for various diseases especially cancer, has been attracted as a pathway suitable for diagnostic purposes. Determination of urinary 8-OHdG is challenging due to its low level within a complex matrix. In this study, a new approach of solid/liquid phase microextraction technique prior to high-performance liquid chromatography diode-array detection (HPLC-DAD) analysis was developed for the determination of trace levels of 8-OHdG in urine samples. The solid/liquid phase microextraction device was constructed by reinforcement of multi-walled carbon nanotubes into the pores of a short segment 2.5 cm of hollow fiber microtube with two ends heat sealed. Based on the optimized procedure, the selected analyte was extracted from an acidic sample solution (10 mL adjusted at pH = 5) into the five extraction devices. After the extraction period (30 min), the 8-OHdG was eluted from the extraction device using methanol (350 µL) under ultrasonication for 5 min. The analytical performance of the method in synthetic urine samples showed good linearity (R2 > 0.999) with the limits of detection of 0.85 ng mL−1, and extraction recovery > 92.36%. The developed microextraction technique exhibited a confident sensitivity, feasible operation, and simplicity in comparison with other published methods and was valid to determinate trace 8-OHdG in urine cancer patients' samples by using a cheap and commonly available HPLC-DAD instrument.
Introduction
8-OHdG, a major product formed by hydroxyl radical attack on the guanine residues of DNA, is considered to be a sensitive and reliable biomarker of oxidative damage in the living cells [1, 2]. The 8-OHdG is excreted in urine without further metabolic changes [3], and the urinary 8-OHdG level in the human body has been examined and correlated with many diseases [4] and used as an indicator for further environmental pollution assessment to other related public health concerns [5].
Elevation level of urinary 8-OHdG in humans is implicated in a number of disorders, including cancer [6, 7], neurodegenerative diseases [8], diabetes [9], Alzheimer's disease [10], cardiomyopathy [11], and cardiovascular or infectious diseases [12]. 8-OHdG has been widely used as a biomarker for cancer diseases such as lung [13], bladder [14], prostate [15], and breast cancers [16], and being lately identified as a biomarker for early detection of leukemia, as leukemia cells begin to multiply in the bone marrow, where cancer begins [17].
Thus, reliable, and quantitative measurement of urinary 8-OHdG is useful in assessing exposure to various carcinogens and crucial for early diagnosis of different diseases to reach a successful therapy and maintain human health.
In particular, the determination of urinary 8-OHdG is confronted by analytical challenges due to the low level of 8-OHdG and the high number of interfering substances in urine samples. Commercially available enzyme-linked immunosorbent assay (ELISA) kits are often used due to their reduced instrumentation, lower costs, and ease of use [18]. However, these assays can show variable performance and may not correlate well with mass spectrometric techniques due to the potential cross-reactivity of 8-OHdG with urea. Liquid chromatography-tandem mass spectrometry (LC-MS/MS) and gas chromatography-mass spectrometry (GC/MS) methods [19, 20] are considered highly sensitive and selective for measuring 8-OHdG concentrations. They can provide accurate results even at low levels. However, these methods often require time-consuming sample preparation procedures such as chemical derivatization or solid-phase extraction (SPE) to remove coexisting substances in urine samples before analysis. These steps are necessary to eliminate potential interferences and enhance the specificity of the analysis. Capillary electrophoresis (CE) and HPLC with electrochemical detection (ECD) [21, 22] methods are other options for quantifying 8-OHdG. However, these methods may not be as sensitive as LC-MS/MS and ECD can have poor stability, affecting the accuracy of the results. The choice of method ultimately depends on the specific requirements of the study, including the desired sensitivity, cost considerations, and the availability of resources and expertise.
An HPLC coupled with an ultraviolet UV system is generally used in most biochemical sample analysis and the instrument is more popular in practice. However, due to the relatively low sensitivity of the UV detector, the HPLC-UV system became difficult to determine the trace 8-OHdG in complex urine matrices [23–25]. Consequently, it is necessary to develop sensitive, efficient, miniaturized, and low-cost sample pretreatment methods to enrich and purify the 8-OHdG prior to the HPLC-UV analysis.
A new mode of hollow fiber miniaturized extraction techniques, namely reinforced hollow fiber solid/liquid phase microextraction (HF-SLPME), has been successfully used for sample pretreatment before injection by chromatography instruments [26, 27]. As an alternative technique of hollow fiber liquid phase microextraction (HF-LPME), the step HF-SLPME device is based on reinforcing the solid adsorbent particles into the pores of the HF-micro-tube to enhance the stability and selectivity of the extraction phase filling the lumen of the HF-micro-tube with the two ends heat-sealed to secure the content [27]. HF-SLPME inherits the common benefits of HF-LPME techniques such as simple operation, low consumption of organic solvent, high enrichment efficiencies, and low analysis cost. In addition, the HF-SLPME procedure provides a high surface area-to-volume ratio by performing several microextraction devices simultaneously, leading to an enhancement of extraction efficiency (EE%), and reducing the time of the experiment and the sample handling process [27].
A great deal of attention has been paid to the application of multi-walled carbon nanotubes (MWCNTs) in various fields of chemical analysis, due to their ability to improve the selectivity of sample preparation procedures [28, 29]. MWCNTs have extraordinary adequacy of structural, good electrical conductivity, high chemical and thermal stabilities with the large available surface area and high adsorption capacity has made them potentially useful in nanotube-reinforced materials, as the sorbents for HF-SLPME technique [30–32].
The purpose of the present study was to develop and optimize a simple and efficient HF-SLPME technique based on the use MWCNTs as reinforced sorbent material in combination with HPLC-DAD to determine the trace level of 8-OHdG in the urine of cancer patients. The potential factors affecting the MWCNTs-HF-SLPME of the analyte were optimized using a univariate approach to obtain the best extraction performance and maximum EE%. Furthermore, comparative studies against referenced standard methods were performed in order to confirm the applicability of the developed methodology.
Experimental
Chemical and materials
The 8-hydroxy-2′-deoxyguanosine was purchased from Biosynth Carbosynth (Compton, England). The acetic acid grades >98%, and hydrochloric acid (37%) were obtained from Sigma-Aldrich (Steinheim, Germany). HPLC-solvents (methanol, acetonitrile, and water) were obtained from Carlo Erba Reagenti (Milan, Italy). Multi-walled carbon nanotubes (MWCNTs) with an outer diameter of 10–20 nm and a length of 5–15 µm according to the specifications reported by the provided manufacturer was purchased from Shenzhen Nanotech Port Co. Ltd. (Shenzhen, China). The hollow fiber microtube (Q3/2 Accurel polypropylene hollow fiber membrane with 600-μm i.d., 200-μm wall thickness, and 0.2-μm pore size) was obtained from Membrana (Wuppertal, Germany).
Characterization of MWCNTs adsorbent
Characterization of MWCNTs was conducted using Bruker vertex 70 IR spectrometer to explore the surface functional groups. Characterization using various adsorption techniques was also conducted, which was previously described [33–36]. This involved determination of methylene blue (MB) relative surface area, determination of basic groups and acidic groups (total acidic groups, phenolic groups, lactonic groups, and carboxylic groups) by Boehm titrations [33–35], and determination of iodine number [33, 34, 36]. A detailed description of the experimental procedures is provided elsewhere [33–36].
Preparation of standard and synthetic urine solution
The stock solution of the 8-OHdG was prepared at a concentration of 1 mg L−1 and stored at 4 °C in the dark. All the used calibration solutions were freshly prepared by the dilution of the appropriate volume of the stock solution with 10 mL of absolute methanol, and for working solutions were diluted with 10 mL of pH = 5 adjusted by using 0.1 M of hydrochloric acid (HCl) and directly subjected to the MWCNTs-HF-SLPME without any further process.
The preparation of synthetic urine samples was based on the formula explained by [37] and was implemented with some modifications. 12.25 g of urea, 1.9 g of potassium chloride, 4.25 g of sodium chloride, 0.59 g of potassium phosphate, 0.32 g of sodium hydroxide 0.23 g of sodium bicarbonate, 0.7 g of creatinine, 0.17 g of ascorbic acid, 0.515 g of citric acid, and 0.14 mL of sulfuric acid into 250 mL ultrapure water under sonicated for 15 min and stored at −20 °C until used.
Patient urine sample collection
In this study, urine samples were collected from three patients diagnosed with cancer. All patients gave their written consent, and the urine samples were collected in the morning. Sample of one male patient, 20 years old, newly diagnosed to have Hodgkin Lymphoma (HL) and treated by doxorubicin, bleomycin, vinblastine, and dacarbazine (ABVD) protocol. Sample for a female patient, 28 years old, diagnosed with stage 1V HL, and started on salvage treatment of ifosfamide, carboplatin, and etoposide (ICE) protocol. Sample of 34 years old female patient diagnosed with acute lymphocytic leukemia, on the active treatment of hyperfractioned cyclophosphamide, vincristine, Adriamycin, and dexamethasone (hyper-cvad) protocol, cycle 5.
MWCNTs-HF-SLPME procedure
A suspension of MWCNTs was prepared by adding 10 mg of MWCNTs to 25 mL of absolute methanol and sonicated for 5 min. The segment of HF (2.5 cm) was rinsed in acetone for 5 min and then immersed in MWCNTs-suspension for 5 min under sonication. After wiping with tissue paper, the lumen of the MWCNTs-HF- micro-tube was filled with 5 μL of methanol using a micro syringe followed by heat-sealed on two ends to secure the content. The MWCNTs-HF-SLPME device was directly immersed in 10 mL of a sample solution (adjusted at pH = 5 using 0.1 M HCl) containing the selected analyte (8-OHdG) with agitation by using a magnetic stirrer bar. After the end of extraction, the MWCNTs-HF-SLPME device was transferred to a micro-vial containing methanol for desorption of the 8-OHdG via ultrasonication and then injected directly into the HPLC-DAD for analysis. To exclude the possibility of memory effect, a newly prepared MWCNTs-HF-SLPME device was used in each experiment.
Instrumentation, chromatographic conditions, and virtual modeling
UHPLC (Ultimate 3000/Dionex, Germering, Germany) system equipped with a binary pump and a UV/DAD detector was employed for the analyses. An Inertsil ODS-3, C18 (150 mm × 4.6 mm × 5 μm) column (Merck Germany) was used for the chromatographic separation. The optimal instrument conditions are injection volume of 10 μL, column temperature at 40 °C, the flow rate at 1 mL min−1, and detection wavelength at 300 nm. The gradient program with methanol as mobile phase A and 0.1% acetic acid in water as mobile phase B is 85% B with a running time of 7.0 min. The retention time of the 8-OHdG was 6.36 min. The Discovery Studio 4.1 was used with the protocol LigandFit Monte-Carlo techniques, and the docked poses were minimized with CHARMm.
Results and discussion
Characteristics of MWCNTs
The FT-IR spectrum of MWCNTs adsorbent is shown in Fig. 1. The following characteristic peaks were reported: C=O stretching peak appeared at 1725 and 1,630 cm−1; O–H stretching appeared as broad peak centered at 3,442 cm−1, the shoulder at 1,386 cm−1 referred to C–OH bending; C–O stretching peaks appeared at 1,118, 1,020 cm−1, the C–H stretching appeared at 2,850 and 2,920 cm−1. By referring to the literature [38], the FT-IR spectrum of MWCNTs showed peaks at 1,410 cm−1 due to OH group; the C=O bands characteristic of carboxyl functional groups (–COOH) was observed at 1711 and 1,638 cm−1, the bands between 1,250 and 950 cm−1 indicated C–O stretching; the –OH band at 3,444 cm−1 was assigned to the associated water. In the adsorption characterization experiments, MWCNTs exhibited the following characteristics: relative surface area as estimated by methylene blue adsorption: 31 m2 g−1; iodine number: 23 mg g−1; total basic groups: 16.7 μmol g−1; total acidic groups 19.8 μmol g−1; phenolic groups: 10.0 μmol g−1; carboxylic groups: 9.8 μmol g−1 [33].
MWCNTs-HF-SLPME optimization
In the present work, MWCNTs-HF-SLPME combined with HPLC–DAD was developed for the extraction and determination of 8-OHdG in urine samples. Based on a univariate approach, the main affecting parameters on the EE% such as the number of MWCNTs-HF-SLPME devices, the pH of the sample solution, the extraction time, and the desorption condition (time of desorption, type, and volume of desorption solvent) were investigated to determine the optimal microextraction procedure. All these parameters were tested in ultrapure water (as a sample solution) and the selection of optimum parameters/values was based on the highest mean for three replicate measurements. Then the analytical performance and applicability of the method were checked in synthetic urine samples spiked with the target analyte.
Effect of sample solution pH
The pH of the sample solution plays an important role in the MWCNTs-HF-SLPME process. The target analyte should be transformed into its neutral molecular forms, which could be achieved by acidifying the sample solution phase. For this purpose, ChemAxon software MarvinView 18.28 was applied to investigate the effect of the pH media (sample solution) ranging from 0 to 9 on the ionization state of the 8-OHdG compound. As shown in Fig. 2, the ionization state of compound 8-OHdG at pH media of 5 mostly (88.05%) exists in unionized form. Experimentally, the pH of the sample solution was investigated in the range of 1–9 with extraction conditions of 10 ng mL−1 of 8-OHdG in 10 mL sample solution, 1 MWCNTs-HF-SLPME device, 60 min extraction time stirring speed 250 rpm, and 150 μL of methanol as desorption solvent for 5 min desorption time. As shown in Fig. 3, the practical results were compatible with the indicated ChemAxon results as the optimum EE% of the 8-OHdG was achieved at pH = 5 and was selected as the pH of the sample solution for further experiments.
Virtually ionization state of compound 8-OHdG at pH = 5 using the ChemAxon software n MarvinView. A, B, and C represent the proposed unionized, positive, and negatively charged forms of compound 8-OHdG at pH = 5, respectively
Citation: Acta Chromatographica 36, 3; 10.1556/1326.2023.01142
The effect of the pH solution on EE% of 8-OHdG by using MWCNTs-HF-SLME-HPLC-DAD. Error bars correspond to standard deviation
Citation: Acta Chromatographica 36, 3; 10.1556/1326.2023.01142
Furthermore, a docking experiment based on using Discovery Studio 4.1 was also used to investigate the expected binding interactions between the unionized 8-OHdG and the prepared device. First, the unionized form of compound 8-OHdG was virtually docked into the model structure of the MWCNTs-HF-SLPME device by using the protocol LigandFit Monte-Carlo techniques. The docked poses were minimized with CHARMm. As shown in Fig. 4, multiple π-π stacking, π-anion, and alkyl interactions were formed between the unionized structures of the 8-OHdG compound and the MWCNTs backbone leading to the high EE% of the selected analyte using the MWCNTs-HF-SLPME device.
Virtually proposed binding interaction between compound 8-OHdG into the Model Structure of MWCNTs-HF-SLPME device
Citation: Acta Chromatographica 36, 3; 10.1556/1326.2023.01142
Effect of the number of MWCNTs-HF-SLPME devices
The surface area of the MWCNTs-HF-SLPME device can influence mass transfer between the acceptor phase (MWCNTs immobilized into the pores of HF-microtube) and the sample solution as well as promote the EE% of analyte [39], thus, the effect of using multiple MWCNTs-HF-SLPME devices on the EE% was evaluated. The MWCNTs-HF-SLPME conditions were set as follows: 10 ng mL−1 concentration of selected analyte, 10 mL of water as a sample solution, 250 rpm stirring speed, 60 min extraction time, 350 µL of methanol as elution solvent sonicated for 5 min. As expected, by increasing the number of MWCNTs-HF-SLPME devices from 1 to 6, the EE% of the 8-OHdG was increased as shown in Fig. 5, however, no additional enhancement was observed when more than 5 devices were used. Therefore, 5 devices were selected for the next optimization steps.
Optimization the number of MWCNTs-HF-SLME devices
Citation: Acta Chromatographica 36, 3; 10.1556/1326.2023.01142
Profile of extraction time
Essentially, the process of the MWCNTs-HF-SLPME is based on partitioning thermodynamics between the donor (sample solution) and acceptor phase as the amount of the analyte extracted is determined by equilibrium time [40], therefore, the EE% is expected to increase with extraction time until reaching of equilibrium. To determine the required time for the establishment of the equilibrium condition in the MWCNTs-HF-SLPME method, the influence of extraction time was investigated by varying the time intervals from 10 to 60 min with 10 ng mL−1 as the concentration of selected analytes and for other conditions (see section effect of the number of MWCNTs-HF-SLPME devices). As observed (Fig. 6), the EE% of the selected analyte increased with time until the MWCNTs-HF-SLPME devices reached equilibrium after 30 min. Hence, 30 min was selected as the optimal time for the next experiments.
Time profile of the proposed method
Citation: Acta Chromatographica 36, 3; 10.1556/1326.2023.01142
Elution conditions
For MWCNTs-HF-SLPME, a solvent is needed for elution of the extracted analytes from the lumen and pores of the HF-microtube. In the present work, after ending the extraction, the MWCNTs-HF-SLPME devices are transferred into a HPLC-micro-vial contained appropriate organic solvent for elution the 8-OHdG via ultrasonication, and for this purpose, elution conditions including the type and volume of elution solvent with the time of sonication must be investigated.
Due to the absence of interfering peaks, HPLC-grade solvents such as acetonitrile, water, and methanol are the most chosen solvents for the elution process in liquid chromatography [41]. The maximum EE% for the selected analytes was obtained when methanol was used as an elution solvent. This result can probably be attributed to the high polarity of methanol with a lower value of dielectric constant [42] and own the potential to break the interactions between the MWCNTs and selected analyte. Thus, methanol was used as an elution solvent for the subsequent experiments.
The volume of elution solvent (methanol) from 150 to 450 μL was also examined. The optimum EE% of the selected analyte is reached by using 350 μL of methanol under ultrasonication while other volumes caused a decreasing in the EE% probably due to insufficiently to immerse the MWCNTs-HF-SLPME devices or as a result of dilution of the analyte, thus, the volume of 350 μL was selected for further process.
Furthermore, a series of ultrasonication times in methanol (1–15 min) was tested. The highest EE% for 8-OHdG is obtained after 5 min of sonication.
Accordingly, the overall results of the MWCNTs-HF-SLPME optimization study, the following experimental conditions were chosen: 5 MWCNTs-HF-SLPME devices, 10 mL of sample solution adjusted at pH = 5 using HCl, 30 min extraction time, 350 μL of methanol as elution solvent for 5 min time of ultrasonication.
Method validation
The method of MWCNTs-HF-SLPME combined with HPLC-DAD for determination of 8-OHdG was assessed by estimating figures of merit including calibration equation, dynamic range, the limits of detection (LOD), and quantification (LOQ) in synthetic urine samples under optimized conditions. The calibration equation derived by plotting peak area against 8-OHdG content expressed as ng mL−1 was: y = 0.0325x + 0.1775. The linear dynamic range was obtained between 2.84 and 250 ng mL−1 with a coefficient of determination R2 = 0.999 (n = 5) of 8-OHdG in synthetic urine samples. The LOD based on 3 times the standard error of intercept divided by coefficients of concentration variable (3 Ser/Cx) [43] was 0.85 ng mL−1, and the LOQ using (10 Ser/Cx) was 2.84 ng mL−1. The determination of precision was made based on the analysis of urine solutions at concentrations of 1, 100, and 250 ng mL−1 (n = 5) on the same day. The proposed method has good repeatability as the relative standard deviations (RSDs) were in the range of 0.36–2.44%.
For the stability test of 8-OHdG in the sample solution, various concentrations (1, 100, and 250 ng mL−1) were assessed under two different conditions. For the first test, the stability of 8-OHdG in synthetic urine solutions were evaluated at the end of each day for 1 week, using the same analysis procedure. The stability test for freeze-thawing, as the second test, was executed for the same stock solutions of 8-OHdG after carrying out five repeated freeze-thaw actions. The results indicate that neither a significant decrease nor degradation was observed in the concentration of 8-OHdG in both tests, and the relative standard deviation (RSD) as listed in Table 1 was less than 2.44% for all samples with extraction recovery >92.36%.
Robustness of the MWCNTs-HF-SLPME-HPLC-DAD method in three different levels
| Level | Spiked concentration ng mL−1 | Mean extracted concentration ng mL−1 (±SD*, n = 3) | Accuracy (Relative error %) | Recovery (%) |
| 1 | 5 | 81.83 (± 1.33) | −0.06 | 93.42 |
| 2 | 5 | 84.21 (± 0.71) | −0.03 | 96.15 |
| 3 | 5 | 80.90 (± 1.42) | −0.07 | 92.36 |
1: The pH = 5.20, extraction time 32 min, for elution methanol volume = 360 μL and ultrasonicated for 6 min.
2: The pH = 5.00, extraction time 30 min, for elution methanol volume = 350 μL and ultrasonicated for 5 min.
3: The pH = 4.80, extraction time 28 min, for elution methanol volume = 340 μL and ultrasonicated for 4 min.
* ± Standard deviation of mean.
The robustness based on the ability of the MWCNTs-HF-SLPME-HPLC-DAD method to remain unaffected against small changes in practical parameters was also evaluated. The results, listed in Table 1, showed negligible differences in the obtained data, and confirmed that the test conditions had no significant effect on the MWCNTs-HF-SLPME-HPLC-DAD analysis results.
Comparison with previous published methods
The data obtained from literature using different kinds of analytical methods for the determination of 8-OHdG in urine samples were selected as references for comparative studies, and the comparative information was presented in Table 2. This result demonstrates that the developed method could extract 8-OHdG efficiently and selectively. As seen in Table 2, our method surpassed some other methods in a wide linear range, low LOD, and rapid analysis. For spectral analysis, more than 10 min is needed for most reference methods, which cannot meet the demand for rapid analysis, while the analysis of the proposed method can be completed within 7 min. Moreover, the proposed method is remarkably easy to use, inexpensive, and environmentally friendly due to the low consumption of organic solvents in both the extraction and elution processes. To summarize, the proposed MWCNTs-HF-SLPME-HPLC-DAD method possessed the advantages of rapid analysis, accuracy, precision, selectivity, and sensitivity.
A Comparison of different methods for urinary 8-OHdG analysis
| Method | Linear range (ng mL−1) | LOD (ng mL−1) | Recovery (%) | Refs. |
| MWCNTs-HF-SLPME-HPLC-DAD | 2.84–250 | 0.85 | 92.3–96.1 | Current study |
| MSPE-HPLC-MSa | 0.05–200 | 0.01 | 82–116 | [44] |
| SPE-LC-MS/MSb | 0.31–10 | 0.31 | 99.4 | [45] |
| SPE-CE-SDc | 10–1,000 | 3 | 82–101.7 | [46] |
| On-line SPE LC-MS/MSd | 0.02–10 | 0.01 | - | [47] |
| PFSPE-HPLC-ECDe | 0.5–50 | 0.058 | 88.8–104.9 | [37] |
| UHPLC-MS/MSf | 0.50–50 | 0.09 | 86.87–104.5 | [48] |
| eVol-MEPS–UHPLC-PDAg | 100–5,000 | 40 | 94.6–103.5 | [49] |
a Magnetic nanoparticles-high performance liquid chromatography-mass spectrometry.
b Solid phase extraction-tandem mass spectrometer.
c Solid-phase extraction -capillary electrophoresis-spectrophotometric detection.
d On-line solid phase extraction-liquid chromatography-tandem mass spectrometry.
e Packed-fiber solid phase extraction high-performance liquid chromatography-electrochemical detection.
f Ultrahigh-performance liquid chromatography-tandem mass spectrometry.
g Digitally controlled microextraction packed sorbent-ultra high-pressure liquid chromatography-photodiodes detection.
Urinary analysis of cancer patients
Finally, the method of MWCNTs-HF-SLPME coupled with HPLC-DAD detection was applied to determine the level of 8-OHdG in urinary samples from a healthy volunteer, and three kinds of cancer patients, respectively. The pH of all urine samples was adjusted to pH = 5 and analyzed without any further treatment. As shown in Fig. 7, the HPLC-DAD chromatograms clearly show the capability of the developed method for separating the excreted 8-OHdG in real urine samples. The quantitative results are listed in Table 3. The 8-OHdG levels of cancer patients were significantly higher than in the healthy person, which indicated higher oxidative damage levels for these patients.
MWCNTs-HF-SLPME-HPLC-DAD chromatograms for urinary samples (A) of healthy volunteers, (B, C, D) of patients diagnosed with Hodgkin Lymphoma, Stage 1V of Hodgkin Lymphoma, and Lymphocytic leukemia, respectively
Citation: Acta Chromatographica 36, 3; 10.1556/1326.2023.01142
Urinary 8-OHdG levels in healthy volunteer, and cancer patients
| Urine sample | 8-OHdG | |
| Peak area (n = 3) | Concentration (ng mL−1) | |
| Healthy volunteer | 0.248 | 2.16 |
| Hodgkin Lymphoma | 1.096 | 28.26 |
| Stage 1V of Hodgkin Lymphoma | 0.477 | 9.21 |
| Lymphocytic leukemia | 2.135 | 60.23 |
Conclusion
In this study, a new MWCNTs-HF-SLPME sample preparation technique was developed for the determination of 8-OHdG a critical oxidative stress biomarker in urine specimens. As a sample matrix, urine was used due to the convenient collection. The developed MWCNTs-HF-SLPME -HPLC-DAD exhibited good EE% and selectivity for 8-OHdG. The MWCNTs-HF-SLPME preparation technique is easier, and simpler with a total analysis period of only 7 min in comparison with other published methods. Since MWCNTs are stable, and environmental-friendly their application as an extract phase avoided complicated synthesis processes and consumption of many toxic organic solvents. Furthermore, the unique structural properties of MWCNTs, including large surface area, high adsorption capacity, and nanoscale dimensions enabled efficient extraction of the target analyte. Additionally, the prepared MWCNTs-HF-SLPME has satisfactory reproducibility at a low cost. All these findings demonstrate that HPLC-DAD, which uses a cheap and commonly available instrument, was valid to determinate trace 8-OHdG in urine cancer patient samples by using the MWCNTs-HF-SLPME technique.
Acknowledgments
The authors would like to acknowledge The Deanship of Scientific Research at The Hashemite University for financial support.
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